Human Induced Pluripotent Stem Cell-Derived Macrophages for Unraveling Human Macrophage Biology

Abstract

Despite a substantial appreciation for the critical role of macrophages in cardiometabolic diseases, understanding of human macrophage biology has been hampered by the lack of reliable and scalable models for cellular and genetic studies. Human induced pluripotent stem cell (iPSC)-derived macrophages (IPSDM), as an unlimited source of subject genotype-specific cells, will undoubtedly play an important role in advancing our understanding of the role of macrophages in human diseases. In this review, we summarize current literature in the differentiation and characterization of IPSDM at phenotypic, functional, and transcriptomic levels. We emphasize the progress in differentiating iPSC to tissue resident macrophages, and in understanding the ontogeny of in vitro differentiated IPSDM that resembles primitive hematopoiesis, rather than adult definitive hematopoiesis. We review the application of IPSDM in modeling both Mendelian genetic disorders and host-pathogen interactions. Finally, we highlighted the potential areas of research using IPSDM in functional validation of coronary artery disease loci in genome-wide association studies, functional genomic analyses, drug testing, and cell therapeutics in cardiovascular diseases.

Keywords: coronary artery disease; genome-wide association study; hematopoiesis; induced pluripotent stem cells; macrophages.

Figure 1. Current status of and future directions for differentiation and application of human IPSDM

Current IPSDM differentiation protocols utilize co-culture, embryoid body- or monolayer-based approaches to produce myeloid progenitors and use M-CSF for directed differentiation to mature macrophages. A number of macrophage functional features have been characterized, including morphology, surface receptor expression, lipid uptake, phagocytosis, cholesterol efflux, CE hydrolysis, cytokine secretion, response to stimuli etc., often in direct comparison to HMDM. Transcriptome landscape, including alternative splicing events and lncRNA profile, also have been analyzed comprehensively. Emerging evidence supports that in vitro IPSDM differentiation represents primitive hematopoiesis rather than adult definitive hematopoiesis. Protocols for iPSC differentiation to human and murine microglial-like cells and murine alveolar macrophages have been implemented while other tissue-specific macrophages and organoid culture for tissue engineering remain to be established. IPSDM have been widely used to model Mendelian disorders and host-pathogen interaction. Additional studies to model functional impact of CAD GWAS loci, epistatic effects, quantitative traits and gene-environment interaction are being pursued. Utilization of IPSDM in genome-wide CRISPR screening to discover novel genes and non-coding transcripts regulating human macrophage function, and identify functional regulatory and causal variants holds great promise. Use of IPSDM in drug testing and IPSDM-based cell therapy are emerging. Improvement in the fidelity, reproducibility and scalability of IPSDM differentiation in conjunction with CRISPR/Cas9 gene editing technologies will further reveal the remarkable potential of IPSDM in multiple areas of functional and translational research in human macrophage biology and pathophysiology. CAD, coronary artery diseases; eQTL, expression quantitative trait loci; hiPSC, human induced pluripotent stem cells; HMDM, human monocyte-derived macrophages; IPSDM, iPSC-derived macrophages; lncRNA, long non-coding RNA; CE, cholesteryl ester. Font in italics denotes potential areas of future research.

Figure 2. Heat map of Spearman’s correlation coefficient (r) for pairwise comparisons of CD14⁺ monocytes, HMDM, iPSC, IPSDM and iPSC-derived myeloid progenitor during an intermediate stage of IPSDM differentiation

RNA-seq data for iPSC, iPSDM and HMDM derived from 3 subjects were downloaded from GSE55536 with 2 lines per subject for iPSC and IPSDM. RNA-seq data for CD14⁺ monocytes from age/race matched subjects were downloaded from GSE87290. We have generated iPSC-derived myeloid progenitors using our published iPSDM differentiation protocol and performed RNA-seq. Those cells were derived from 2 subjects with one line per subject, who are among the 3 subjects described in GSE55536. The iPSC-derived myeloid progenitors are CD43⁺CD34⁺CD45⁺CD18⁺ and are harvested at day-15 of the differentiation from suspension culture of EBs and then differentiated to mature macrophages using serum and M-CSF in adherent culture for 7 days. Depth coverage among samples were normalized as described by Anders and Huber. Genes expressed at FPKM > 1% expression of all genes were included for each sample. FPKM values were then normalized across samples. Spearman’s correlation coefficient was calculated for each pair of samples and visualized by heat map. The transcriptome of iPSC-myeloid progenitors are highly correlated with that of IPSDM, but to a lesser extent with that of CD14⁺ monocytes, supporting their identity as “macrophage precursors” during a progressive commitment and maturation of macrophages, rather than “monocytes” as suggested also by Haenseler et al. using a different differentiation protocol to obtain IPSDM. HMDM, human monocyte-derived macrophages; IPSDM, iPSC-derived macrophages.